1. Competing Pathways and Multiple Folding Nuclei in a Large Multidomain Protein, Luciferase 
Zackary N. Scholl, Weitao Yang, Piotr E. Marszalek 
Biophysical Journal 
Volume 112, Issue 9, Pages (May 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
Schematic of the refolding experiments for I913-Luciferase-I914 construct and I919. The distance between the substrate and the tip is modulated by the piezo, which executes a constant velocity ramp as shown in (A). The force on the molecule as determined by the extension, closely approximated by a wormlike chain, is shown in (B). The shaded region in (A) and (B) is where the molecule experiences a force that is below the typical isometric unfolding force (5 pN). The physical schematic of the experiment is shown in (C), where the piezo moves toward the cantilever, at which a protein can fold (denoted by the star), which is determined by the subsequent unfolding that results in a force peak.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. Figure 2
Four examples of successive refolding attempts on a single molecule of I913-Luciferase-I914. The order of recordings goes from bottom to top for each set. As can be seen, the I91 molecules (200 pN unfolding force) are able to refold successfully upon each attempt. A single non-I91 peak appears a fraction of the time, and a wormlike chain (dashed line) shows a contour-length increments that match previously reported contour-length increments for individual domains of Luciferase.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
Distributions of the contour-length increments for non-I91 peaks in refolding recordings (peaks with force <120 pN; see Fig. S9 for all peaks). Top shows the distribution for refolding of I919. Bottom shows the distribution for the refolding of I913-Luciferase-I914, which has events not present in I919 and match previous results for the unfolding of full Luciferase. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

5. Figure 4
Representation of the main folding routes from coarse-grain simulations (back arrows representing unfolding not shown; for complete Markov state model, see Fig. S2). The numbers above each state represent that state number for the 14-state clustering (reference in Fig. S3), while the percentage is the probability of obtaining that structure during any given folding pathway (see Materials and Methods). The arrow thickness qualitatively represents the relative flux through the pair of states. Structures with a star indicate that they were evaluated experimentally by SMFS. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
Representative structures of the topologically misfolded states obtained during folding of Luciferase, with the state labeled in bold (referring to 14-state clustering in Fig. S2). The structures are colored from red (N-terminal residues) to blue (C-terminal residues). The right side shows a template molecule of the correctly folded form of Luciferase. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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7. Figure 6
Three representative examples of experimental force-spectroscopy recordings obtained for each construct in this study. All the vertical scale bars represent 100 pN and the horizontal scale bars all represent 50 nm. Arrows indicate the force rupture event used in analysis.
Biophysical Journal  , DOI: ( /j.bpj )
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8. Figure 7
Experimental SMFS results of the contour-length increment (left) and unfolding force (right) for Luciferase truncations. Previous results on the full Luciferase (47) are shown at the top for comparison, which shows unfolding of C-terminal domain (light shading), middle domain (dark shading), and N-terminal domain (solid). The dashed bar shows the result for the contour-length increment calculated from SMD simulations.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions